ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales , including Klebsiella pneumoniae , Salmonella enterica , and Yersinia pestis . As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery.

Genomic diversity and organization of complex polysaccharide biosynthesis clusters in the genus Dickeya

The pectinolytic genus Dickeya (formerly Erwinia chrysanthemi) comprises numerous pathogenic species which cause diseases in various crops and ornamental plants across the globe. Their pathogenicity is governed by complex multi-factorial processes of adaptive virulence gene regulation. Extracellular polysaccharides and lipopolysaccharides present on bacterial envelope surface play a significant role in the virulence of phytopathogenic bacteria. However, very little is known about the genomic location, diversity, and organization of the polysaccharide and lipopolysaccharide biosynthetic gene clusters in Dickeya. In the present study, we report the diversity and structural organization of the group 4 capsule (G4C)/O-antigen capsule, putative O-antigen lipopolysaccharide, enterobacterial common antigen, and core lipopolysaccharide biosynthesis clusters from 54 Dickeya strains. The presence of these clusters suggests that Dickeya has both capsule and lipopolysaccharide carrying O-antigen to their external surface. These gene clusters are key regulatory components in the composition and structure of the outer surface of Dickeya. The O-antigen capsule/group 4 capsule (G4C) coding region shows a variation in gene content and organization. Based on nucleotide sequence homology in these Dickeya strains, two distinct groups, G4C group I and G4C group II, exist. However, comparatively less variation is observed in the putative O-antigen lipopolysaccharide cluster in Dickeya spp. except for in Dickeya zeae. Also, enterobacterial common antigen and core lipopolysaccharide biosynthesis clusters are present mostly as conserved genomic regions. The variation in the O-antigen capsule and putative O-antigen lipopolysaccharide coding region in relation to their phylogeny suggests a role of multiple horizontal gene transfer (HGT) events. These multiple HGT processes might have been manifested into the current heterogeneity of O-antigen capsules and O-antigen lipopolysaccharides in Dickeya strains during its evolution.

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Polysaccharide co-polymerase WzzB/WzzE chimeras reveal transmembrane 2 region of WzzB is important for interaction with WzyB

The ability of bacteria to synthesise complex polysaccharide chains at a controlled number of repeating units has wide implications for a range of biological activities that include: symbiosis, biofilm formation and immune system avoidance. Complex polysaccharide chains such as the O antigen (Oag) component of lipopolysaccharide and the enterobacterial common antigen (ECA) are synthesised by the most common polysaccharide synthesis pathway used in bacteria, known as the Wzy-dependent pathway. The Oag and ECA are polymerized into chains via the inner membrane proteins WzyB and WzyE, respectively, while the respective co-polymerases WzzB and WzzE modulate the number of repeat units in the chains or “the modal length” of the polysaccharide via a hypothesised interaction. Our data shows for the first time “cross-talk” between Oag and ECA synthesis in that WzzE is able to partially regulate Oag modal length via a potential interaction with WzyB. To investigate this, one or both of the transmembrane regions (TM1 and TM2) of WzzE and WzzB were swapped creating six chimera proteins. Several chimeric proteins showed significant increases Oag modal length control, while others reduced control. Additionally, co-purification experiments show an interaction between WzyB and WzzB for the first time without the use of a chemical cross-linker, and a novel interaction between WzyB and WzzE. These results suggest the TM2 region of Wzz proteins plays a critical role in Oag and ECA modal length control, presumably via the interaction with respective Wzy proteins, thus providing insight into the complex mechanism underlying the control of polysaccharide biosynthesis. Importance Bacteria synthesise complex polysaccharide chains at a controlled number of repeating units, this has wide implications for a range of bacterial activities involved in virulence. Examples of complex polysaccharide chains include, the O antigen (Oag) component of lipopolysaccharide and the enterobacterial common antigen (ECA), both of these examples are predominantly synthesised by their own independent Wzy-dependent pathway. Our data show for the first time “cross-talk” between Oag and ECA synthesis and identifies novel physical protein-protein interactions between proteins in these systems. These findings further the understanding of how the system functions to control polysaccharide chain length which has great implications for novel biotechnologies and/or the combat of bacterial diseases.

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Here, we used the WHO recommended H1N1 antigens (2019-2020 season) in a systematic analysis to demonstrate that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, thereby preserving the antigens. The purified inactivated virions with higher NA content show a more spherical morphology, a different balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair that can be utilized to produce viral-based influenza vaccines with increased NA content which can elicit more balanced inhibitory antibody responses to NA and HA.